AU2007310738B2 - Method for in vitro diagnosis of PVL-producing Staphylococcus aureus - Google Patents

Abstract

The present invention relates to a method for

Description

1 Method for in vitro diagnosis of PVL-producing Staphylococcus aureus The present invention relates to the field of infections associated with the presence of bacteria belonging to the Staphylococcus genus and of the species Staphylococcus aureus, which produce Panton-Valentine Leukocidin (PVL). More 5 particularly, the subject of the present invention is a method for determining PVL producing Staphylococcus aureus using a routine immunological test in a biological sample that may contain Staphylococcus aureus. Infections with PVL-producing Staphylococcus aureus are essentially responsible for community infections. Staphylococcus aureus expresses a large variety 10 of virulence factors, among which is Panton-Valentine Leukocidin (PVL), a cytotoxin which promotes tissue damage'. PVL is a toxin commonly present in community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains which are spreading worldwide 2 . PVL, and also other leukocidins, is a protein belonging to the family of 15 synergohymenotropic toxins. All the toxins of this family are constituted of two polypeptide components, with synergic action, denoted S and F. PVL is encoded by two cotranscribed contiguous genes, lukF-PV and lukS-PV. PVL is an exotoxin since it is excreted into the extracellular medium whether or not there has been lysis of the bacterium, unlike endotoxins or lipopolysaccharides, which, themselves, are released 20 only when the Gram- bacterium which secretes them has been destroyed. PVL-producing Staphylococcus aureus, which are Gram+ bacteria, are associated with specific human infections such as cutaneous infections or subcutaneous infections, of the boil, abscess, cellulitis and myositis type, osteoarticular infections, and also severe necrotizing pneumonias which affect mainly children and young adults 25 with a mortality rate of approximately 70%. The pathogenesis is not completely known, but several lines of evidence suggest that PVL plays an important role in the physiopathology of infections with PVL producing Staphylococcus aureus: i) the strong epidemiological link with isolates of S. aureus which 30 synthesize PVL and the clinical presentation of the infection, 2 ii) the high frequency of leukopenia, a known effect of PVL, iii) the necrotic lesions of the respiratory tract, resembling the necrosis induced by intradermal injection of PVL in rabbits, iv) the presence of PVL in the lungs targeting the polymorphonuclear cells, 5 v) only PVL-producing strains or purified PVL induce necrotizing pneumonia in experimental models 7 . PVL-producing S. aureus are currently detected using tests for detecting nucleic acids, and in particular by detecting the lukF-PV and lukS-PV genes by PCR 3 . Such tests have the drawbacks that they are indirect since they do not make it 10 possible to confirm that the gene is functional and/or expressed, they are expensive owing to the need for specific equipment, they are not very fast and they are difficult to carry out. In addition, in the context of the use of a PCR, contamination problems may occur. The detection of PVL has also been carried out, from time to time, by 15 immunodiffusion using polyclonal antibodies 4 , but this technique was quickly abandoned in favor of the detection of genes since it is not very easy to carry out and difficult to standardize, such that it does not constitute a good tool for routine use. Owing to the seriousness of certain infections due to PVL-producing Staphylococcus aureus, it is becoming urgent to have available a rapid test that is 20 simple to carry out and that would overcome the drawbacks of the tests currently used to detect PVL-producing S. aureus. Patent application EP 597 110 A describes the fact that the detection of MRSA can be carried out by means of routine immunological tests using anti-PVL monoclonal antibodies. However, although these tests make it possible to overcome the above 25 drawbacks, the specificity of detection is not sufficient. Against all expectations, the applicants have now demonstrated that the specificity of the detection of PVL-producing S. aureus using a routine immunological test can be improved by carrying out a pretreatment of the biological sample in order to denature the PVL, although it is known to those skilled in the art that, like any 30 exotoxin, PVL is sensitive to physicochemical agents such as temperature'.

Thus, the subject of the present invention is a method for in vitro diagnosis of Staptylococcus aureus producing Panton-Valentine Leukocidin (PVL) using a biological sample derived from an individual liable to be colonized by or infected with Staphylococcus aureus wherein the diagnosis is carried out by detection of PVI. using a routine immunological test., characterized in that said biological sample is preheated in order to denature the PVI,. According to one aspect, the present invention provides a method for in vitro diagnosis of Staphylococcus aureus producing Panton-Valentine Leukocidin (PVL) using a biological sample derived from an individual liable to be colonized by or infected with Staphylococcus aureus, wherein the diagnosis is carried out by detection of PVL using a routine immunological test chosen from an enzyme-linked immunosorbent assay (ELISA), lateral flow immunochromatography and an agglutination test and wherein said biological sample is pretreated in order to denature the PVl, the pretreatment composing heating at a temperature of between 60 and I00"C for a period of at least 10 min or using an acid. The expression "PV L-producing Staphvlococcus aureus" is intended to mean all the Staphylococcus aureus strains capable of producing PVL. i.e. both the methicillin-sensitive strains (also known as MSSA), and those which are methicillin-resistant (also known as MRSA). The expression "individual liable to be colonized by Staphvlococcus aureus" is intended to mean the individuals at risk who may be healthy carriers of this bacterium, such as hospital staff The expression "individual liable to be infected with Staphylococcus aureus " is intended to mean the patients displaying the symptoms of an infection with Staphylococcus aureus. The expression "infection with Staphylococcus aureus" is intended to mean any infection caused by the presence of this bacterium in an organism, whether these infections are with PVL-producing Staphylococcus aureus or with Staphylococcus aureus which do not have such a capacity By way of example of such infections, mention may be made of suppurative infections of the skin and of the soft tissues, respiratory tract infections, central nervous system infections, urinary infections, cardiac valve and endovascular infections and muscle and bone infections. Such infections and the associated symptoms are largely known to those skilled in the art and are described, in particular, in the Pr6cis de Bact6riologie Clinique' [Handbook of Clinical Bacteiology).

3a The expression "bioJogical sample derived from an individual liable to be colonized by or infected with Staphylococcus aureus" is intended to mean any sample liable to contain these bacteria or else the excreted PVI., such as pus, a respiratory sample, a nasal sample, urine or a hemoculture. The biological sample used in the method of the invention may be the 0:'WPw S.iOn py 4 unmodified sample, or else it may consist in a culture of the bacteria that is derived from this sample. The culture may be carried out on a solid medium, such as a Petri dish, or in a culture broth, it being understood that the culture broth can be inoculated with said sample or else with colonies of Staphylococcus aureus isolated beforehand 5 from said biological sample by methods well known to those skilled in the art, such as by seeding on a Petri dish. The immunological test will then be carried out either directly on the solid medium, or in the culture broth. In general, the culturing of the bacteria of interest or of said sample lasts approximately 24 h. It may be noted that, whatever the culture, the culture medium comprises an agent for promoting the 10 production of PVL, such as the CCY medium (casein hydrolyzate and yeast extract medium), with or without the use of molecules enabling a greater production of PVL, such as oxacillin and bactracin at a sub-inhibitory dose. The term "immunological test" is intended to mean any immunological test using a binding partner capable of binding specifically to PVL, whether it is the LukF 15 fraction of PVL, the LukS fraction of PVL, or else both fractions simultaneously. The term "routine immunological test" is intended to mean any immunological test widely used in the routine practice of a laboratory. By way of example of routine immunological tests, mention may be made of sandwich tests of ELISA or immunochromatographic (also known as lateral flow) type, and particle, for example 20 polystyrene particle, agglutination tests. All of these tests are widely known to those skilled in the art. The sandwich test can be carried out in one or more steps, i.e. without a washing step or with one or more washing steps. . By way of example of a PVL-specific binding partner, mention may be made of antibodies, antibody fractions, receptors, mimotopes and any other molecule capable of 25 binding to PVL. The binding-partner antibodies are, for example, either polyclonal antibodies or monoclonal antibodies. The polyclonal antibodies can be obtained by immunization of an animal with PVL, a PVL fraction or a PVL peptide, followed by recovery of the serum of said 30 animal. The antibodies can be used purified or nonpurified, in the method of the 5 invention. The purification of the polyclonal antibodies can be carried out, for example, by taking the serum of said animal and separating said antibodies from the other serum constituents, in particular by affinity chromatography on a column to which an antigen specifically recognized by the antibodies, in particular PVL, is attached. 5 The monoclonal antibodies can be obtained by the hybridoma technique, the general principle of which is summarized hereinafter. In a first step, an animal, generally a mouse (or cells in culture in the context of in vitro immunization), is immunized with PVL, a PVL fraction or a PVL peptide, and the B lymphocytes of this mouse are then capable of producing antibodies against said 10 antigen. These antibody-producing lymphocytes are subsequently fused with "immortal" myeloma cells (murines in the example) so as to produce hybridomas. The heterogeneous mixture of the cells thus obtained is then used to form a selection of the cells capable of producing a particular antibody and of multiplying indefinitely. Each hybridoma is multiplied in the form of a clone, each one resulting in the production of a 15 monoclonal antibody specifically recognizing PVL. The antibodies may be used purified or nonpurified in the method of the invention. The monoclonal antibodies may be purified in particular according to the affinity chromatography technique described above. The monoclonal antibodies may also be recombinant antibodies obtained by 20 genetic engineering, using techniques well known to those skilled in the art. The expression "antibody fragments useful for the purposes of the invention" is intended to mean fragments of F(ab')2, Fab, Fab' or scFv type, of a native antibody, which have kept the capacity of specific binding to PVL. The method of the invention is preferably carried out with the following 25 characteristics, taken alone or in combination: - the routine immunological test is a sandwich method, , - it uses LukS-PV- or LukF-PV-specific binding partner(s), - it uses antibody fragments, and in particular the F(ab')2 fragment. In the context of a sandwich test using two PVL-specific binding partners, use 30 may, for example, be made of two monoclonal antibodies, two polyclonal antibodies, or 6 fragments thereof, or else one monoclonal antibody and one polyclonal antibody, or fragments thereof In the context of a particle agglutination test, a single PVL-specific binding partner is used, for example either a monoclonal antibody or a polyclonal antibody, which may or may not be in the form of a fragment. 5 According to one particular embodiment, the immunological test uses at least one anti-PVL monoclonal antibody. In the context of a particle agglutination test, the PVL-specific binding partner is used in capture mode. In the context of a sandwich test, they are used in capture mode and in detection mode. 10 When the binding partner is used as a detection reagent, it is labeled in order to reveal the PVL/binding partner binding. The term "labeling of binding partners" is intended to mean the attachment of a label capable of directly or indirectly generating a detectable signal. A nonlimiting list of these labels comprises: 15 e enzymes which produce a signal detectable, for example, by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, a-galactosidase or glucose-6-phosphate dehydrogenase, e chromophores, such as fluorescent, luminescent or dye compounds, * radioactive molecules, such as 32 P, "S or 121, and 20 e fluorescent molecules, such as alexa or phycocyanins. Indirect detection systems can also be used, for instance ligands capable of reacting with an anti-ligand. The ligand/anti-ligand pairs are well known to those skilled in the art, this being the case, for example, of the following pairs: biotin/streptavidin, hapten/antibody, antigen/antibody, peptide/antibody, sugar/lectin, 25 polynucleotide/sequence complementary to the polynucleotide. In this case, it is the ligand which carries the binding partner. The anti-ligand may be detectable directly via the labels described in the preceding paragraph, or may itself be detectable via a ligand/anti-ligand. These indirect detection systems may result, under certain conditions, in an 30 amplification of the signal. This signal amplification technique is well known to those 7 skilled in the art, and reference may be made to the article in J. Histochem. Cytochem 6 . 45: 481-491, 1997. Depending on the type of labeling used, those skilled in the art will add reagents for visualizing the labeling. 5 In the case of "competition" methods, the PVL is labeled as described above for the binding partner. The PVL-specific binding partner, if it is used in the capture mode, is then directly or indirectly immobilized on a solid phase using methods known to those skilled in the art. 10 The pretreatment of the sample used in the method of the invention may be any protein-denaturing treatment widely known to those skilled in the art. This treatment may be carried out, for example, by modification of the pH, by using chemical denaturing agents, such as urea, sodium dodecyl sulfate (SDS) or guanidinium ions, or else by heating the sample which contains the protein to be denatured. 15 According to one embodiment, the pretreatment of said sample consists in heating at a temperature of between 60 and I 00*C for a period of at least 10 min. Preferably, the heating is carried out at a temperature of between 80 and 1 00*C, more preferably between 90 and I 00 0 C. Of course, the pretreatment of the sample will be applied to the sample itself 20 when the routine immunological test is carried out in said sample, or else it will be applied to the culture when the immunological test is carried out in said culture. The method of the invention may also comprise an additional step of verifying the presence of Staphylococcus aureus in said biological sample, it being possible for this step to be carried out beforehand or concomitantly. The methods for detecting these 25 bacteria are known to those skilled in the art, and mention may be made, by way of example, of the use of media containing a chromogenic agent specific for this bacterium, as described, for example, in patent application WO 02/079486 filed by one of the applicants. Similarly, the method of the invention may comprise an additional step 30 consisting in determining whether the Staphylococcus aureus present in said biological 8 sample are methicillin-resistant (MRSA) or methicillin-sensitive (MSSA) bacteria, thereby constituting a particular embodiment of the invention. This step of determining the methicillin sensitivity (MRSA or MSSA) can be carried out by methods widely known to those skilled in the art, such as immunological 5 tests using, for example, a binding partner specific to the PBP2' protein, which is a protein expressed only by MRSA, tests for detecting nucleic acid or else microbiological tests, for example using a medium containing an antibiotic such as oxacillin or a cephalosporin, for example cefoxitin. The invention will be understood more clearly by means of the following 10 examples given by way of nonlimiting illustration, and also by means of the attached figures 1 to 3, in which: - figure 1 is a graph giving the results of an ELISA test (OD of each strain) for the detection of PVL in biological samples containing strains of Staphylococcus aureus producing PVL (PVL+) or not producing PVL (PVL-), 15 - figure 2 is a graph giving the results of an ELISA test (OD of each strain) for the detection of PVL in biological samples containing strains of Staphylococcus aureus producing PVL (PVL+) or not producing PVL (PVL-), said sample having been pretreated by heating in order to denature the PVL, and - figure 3 is a graph giving the results of an ELISA test (OD of each strain) for the 20 detection of PVL in biological samples containing strains of Staphylococcus aureus producing PVL (LY990084, A92007, LUG855) or not producing PVL (LY990333, LY991321, RN69 11), said sample having been pretreated by heating (treatment 1), pretreated using a chemical denaturing agent (treatment 2), or pretreated using a denaturing agent and by heating (treatment 3), state 0 corresponding to no pretreatment 25 of the sample. Example 1: Preparation of anti-PVL antibodies 1. Production of recombinant PVL The LukS His-Tag and LukF His-Tag proteins of respective sequence 30 SEQ ID No. I and SEQ ID No. 2 were produced using the vector pIVEX 2.4d (Roche) 9 used to transform a strain of Escherichia coli BL21star(DE3) pLys (Invitrogen) during culturing of 2 liters at 37 0 C with shaking, induction for 3 h at 37*C with 1mM IPTG (isopropyl-beta-D-thiogalactopyranoside, Eurobio), and then culturing for a further 5 hours. The culture is dispensed into 6 different tubes. 5 Each cell pellet is recovered by centrifugation at 4000 g for 20 min and removal of the supernatant. The recombinant proteins are purified using the QlAexpressionist kit according to the recommendations of the supplier (Qiagen). The pellet is lyzed with 20 ml of a nondenaturing lysis solution (Qiagen) at 4*C, and then conserved overnight at -80*C. After thawing, the lysis is continued using a treatment with lysozyme 10 (Eurobio) 1 mg/ml for I h at 4'C, before sonication using a Vibracell (Bioblock) at 100 watt, for 2 min in cycles of 6 s. The solution is centrifuged at 10 000 g for 30 min at 4*C. The supernatant is brought into contact with 5 ml of agarose-NiNTA (Qiagen) for 3 h at 4*C with gentle circular agitation. The agarose is then placed in a 20 ml column (Biorad). The column 15 is washed with 200 ml of wash buffer (Qiagen), and then eluted with 250 mM imidazole as eluting buffer. This purification is completed by purification on a monoSP ion exchange column (Amersham). The recombinant proteins are concentrated on a Centricon (Vivaspin), dialyzed against a 50 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer, and then eluted 20 with a gradient of NaCl (0 to I M). The proteins are then dialyzed against apyrogenic sterile water. 2. Production of monoclonal antibodies The following monoclonal antibodies were obtained: - anti-LukF: IODIA10, 16A10A3, 6H10 E5 and 25 - anti-LukS: 2H2H12, 3D9D12, 7CIF9, 7F8D7, 18A4E10 as follows: Ant-Luk F monoclonal antibodies: 10DJA10 , 16A10A3 , 6H1OE5. The mice were immunized according to the following protocol: at day DO, intraperitoneal injection of 10 ptg of recombinant Luk F protein in the presence of 30 complete Freund's adjuvant. At days D14, D28, further intraperitoneal injection of the 10 same amount of recombinant Luk F protein in the presence of incomplete Freund's adjuvant. Four days before the fusion, an intravenous injection of 50 pg of Luk F antigen diluted in physiological saline is given. 1600 supernatants were screened by the indirect ELISA technique. The 5 plates were "coated" with 100 pl of antigen (recombinant Luk F protein) at I pg/ml in PBS buffer, pH 7.2. The "coated" plates were incubated overnight at the temperature of 18-22*C. The plates were saturated with 200 pl of PBS-l% milk and subjected to incubation for 1 hour at 37*+/-2*C. 100 p1 of supematants or of ascites fluid diluted in PBS buffer-0.05% tween 20, were added and the plates were incubated for 1 hour at 10 37'+/-2*C. 100 pl of goat anti-mouse Ig(H+L) polyclonal antibody conjugated to alkaline phosphatase (Jackson Immunoresearch ref: 115-055-062), diluted to 1/2000 in PBS buffer-I% BSA, were added and the plates were subsequently incubated for 1 hour at 37'+/-2*C. 100 pl of PNPP (bioMrieux ref. 60002990) at the concentration of 2 mg/ml in DEA-HCI (bioMrieux ref. 60002989), pH=9.8, were added. The plates 15 were subjected to incubation for 30 minutes at 37*+/-2*C. The reaction was blocked by adding 100 pl of IN NaOH. Three washes are carried out between each step, with 300 g1 of PBS-0.05% tween 20. A further wash in distilled water is carried out before adding the PNPP. 72 supernatants were found to be positive by indirect ELISA with an OD > 0.6. 20 After the specificity test, the abovementioned three antibodies are produced. Anti-Luk S monoclonal antibodies: 2H2H12, 3D9D12, 7C1F9, 7F8D7, 18A4E10 The mice were immunized according to the following protocol: at day DO, intraperitoneal injection of 10 pg of recombinant Luk S protein in the presence of complete Freund's adjuvant. At days D14, D28, a further intraperitoneal injection of 25 the same amount of recombinant LuK S protein in the presence of incomplete Freund's adjuvant. Four days before the fusion, an intravenous injection of 50 pg of recombinant Luk S protein, diluted in physiological saline, is given. 1800 supernatants were screened by the indirect ELISA technique. The plates were "coated" with 100 pl of recombinant Luk S protein at 1 pg/ml in PBS buffer, 30 pH 7.2. The "coated" plates were incubated overnight at a temperature of 18-22*C. The 11 plates were saturated with 200 pl of PBS-l% milk and subjected to incubation for 1 hour at 37*+/-2*C. 100 pl of supernatants or of ascites fluid, diluted in PBS buffer 0.05% tween 20, were added and the plates were incubated for 1 hour at 37*+/-2*C. 100 pl of goat anti-mouse Ig(H+L) polyclonal antibody conjugated to alkaline 5 phosphatase (Jackson Immunoresearch ref.: 115-055-062), diluted to 1/2000 in PBS buffer-I% BSA, were added and the plates were then incubated for 1 hour at 37*+/-2*C. 100 pl of PNPP (Biom6rieux ref. 60002990) at the concentration of 2 mg/ml in DEA-HCl (Biom6rieux ref. 60002989), pH=9.8, were added. The plates were subjected to incubation for 30 minutes at 37*+/-2*C. The reaction was blocked by 10 adding 100 l1 of IN NaOH. Three washes are carried out between each step, with 300 p1 of PBS-0.05% tween 20. A further wash in distilled water is carried out before adding the PNPP. 51 supernatants were found to be positive by indirect ELISA with an OD > 0.6. After the specificity tests, the abovementioned five antibodies are produced. 15 3. Production of polyclonal antibodies 3.1. Production of anti-Luk S polyclonal antibodies The anti-LukS polyclonal antibodies No. 173/89 and 176/89 were obtained as follows: At day DO, rabbits (New Zealand White) received an intradermal injection of 20 200 pg of LukS-PV synthetic peptide of sequence CSGHDPNLFVGYKPYSQN (SEQ ID No.3), N-terminal coupled with KLH (Keyhole Limpet Hemocyanin) (Agro-Bio). At days D14, D28, D42 and D81, the rabbits received a further subcutaneous injection of the same amount of synthetic peptide coupled to KLH. The serum of the animals is taken at DO, D49 and D89. The rabbit sera taken at D89 are purified by affinity 25 chromatography on said synthetic peptide. For this, said peptide is coupled onto a 1 ml Hi-Trap sepharose column according to the recommendations of the manufacturer (Amersham-Pharmacia). The serum is diluted 50/50 in PBS buffer, pH 7.4, and then injected onto the column at a rate of I ml/min. After washing with PBS, pH 7.4, and then elution with 50 mM of glycine/HCl mixture, pH 3, the eluted fractions are 30 immediately neutralized, and then dialyzed against 0.15 M PBS, pH 7.4.

12 3.2. Production of anti-Luk F polyclonal antibodies The anti-LukF polyclonal antibodies were obtained as follows: At day DO, rabbits (New Zealand White) received an intradermal injection of 200 ptg of LukF-PV synthetic peptide of sequence CNFNWIGNNYKDENRATHTS 5 (SEQ ID No.4), N terminal coupled with KLH (Keyhole Limpet Hemocyanin) (Agro Bio). At days D14, D28, D42 and D81, the rabbits received a further subcutaneous injection of the same amount of synthetic peptide coupled to KLH. The serum of the animals is taken at DO, D49 and D89. The rabbit sera taken at D89 are purified by affinity chromatography on said synthetic peptide. For this, said peptide is coupled onto 10 a I ml Hi-Trap sepharose column according to the recommendations of the manufacturer (Amersham-Pharmacia). The serum is diluted 50/50 in PBS buffer, pH 7.4, and then injected onto the column at a rate of I ml/min. After washing with PBS, pH 7.4, and then elution with 50 mM of glycine/HCl mixture, pH 3, the eluted fractions are immediately neutralized, and then dialyzed against 0.15M PBS, pH 7.4. 15 4. Production and coupling of antibody fragments The anti-LukS polyclonal antibodies 173/89 and 176/89 were digested with pepsin bound to agarose, for I h 30 at pH 3.5 at 37*C, in order to eliminate the Fc fragment thereof and thus to obtain the F(ab')2 fragments. These fragments were labeled with peroxidase as follows: the F(ab')2 fragments 20 are dialyzed against carbonate buffer, pH 9.6, and coupled to peroxidase (Roche) oxidized beforehand with NaIO 4 , for 2 hours at 18-25*C with a ratio of: 1 mol of F(ab')2 per 2 mol of peroxidase. The coupling is then blocked with NaBH 4 for 1 hour at 2-8*C, and the product is then dialyzed against PBS buffer with preservatives. They were subsequently labeled with biotin as follows: the F(ab')2 fragments 25 are dialyzed against carbonate buffer, pH 8.3, and coupled to biotin-NHS (Roche) for 1 hour at 18-25*C with a ratio of I mol of F(ab')2 per 10 mol of biotin. The coupling is blocked with lysine for 20 minutes at 18-25'C, and the product is then dialyzed against PBS + azide. 30 Example 2: Diagnosis of PVL-producing Staphylococcus aureus strains 13 1. Preparation of the biological sample The clinical strains of Staphylococcus aureus are precultured on GP agar (Difco: 10 g/l peptone, 5 g/l yeast extract, 17 g/l agar; Sigma: 5 g/l NaCl, I g/l glucose). After 18 h at 37*C, the purity is verified before inoculating some colonies (approximately 5 107 CFU/ml) into 5 ml of CCY medium (Difco: 30 g/l yeast extract, 20 g/l casamino acid; Sigma: 3.11 g/l Na 2

HPO

4 2H 2 0, 0.41 g/l KH 2

PO

4 , 23 g/l pyruvic acid) in a 25 ml glass Erlenmeyer flask. After culturing for 18 h at 37*C with shaking, the culture supernatants are recovered after centrifugation (8000 g, 4*C, 10 min), and are then conserved for a short period at 4*C or at -20*C before use. 10 Each strain was given an identifier (A or LY plus 6 numbers) and is denoted "+ " if it produces PVL or" -" if it does not produce PVL. 2. ELISA test The ELISA plate wells (Greiner, 100 pl/well) are coated with anti-LukS antibody 18A4E10 by incubation with a solution of this antibody diluted to 10 ig/ml in 15 PBS (Sigma PBS, pH 7.4; 0.1% sodium azide) for 18 h at ambient temperature (approximately 22'C). After 5 washes in PBS Tween 20 (Biorad ImmunoWash 1575 machine), the plate is saturated with a solution of PBS Tween (0.05%)/10% milk/O.5% BSA (Sigma) for 2 h at ambient temperature (150 pl/well). After 5 washes with PBS Tween (0.05%), the culture supernatants are incubated 20 for 75 min at 37*C. After 5 washes with PBS Tween (0.05%), the solution of rabbit antibody 176 89, F(ab'2) fragment labeled with peroxidase, diluted to 1/100 in PBS-T 5% milk, is incubated for 75 min at 37*C. After 5 washes with PBS-Tween, 75 Pl of TMB solution (3,3',5,5'-tetramethylbenzine substrate, Sigma) are added and the plate is incubated for 25 30 min in the dark. The reaction is stopped by adding 75 pl of IN H 2

SO

4 . The plates are read (OD) at 450 nm using the MicroPlate MP Reader 680 (Biorad). 3. Results The results are shown in figure 1, which is a graph giving the OD of each strain. The results show that, among the 15 PVL+ strains, 12 strains are clearly 30 detected and that, among the 14 PVL- strains, 3 false-positive results are observed.

14 These results clearly show that it is possible to differentiate between the PVL+ strains and the PVL- strains using a routine immunological test, but that the latter lacks specificity. 5 Example 3: Diagnosis of PVL-producing Staphylococcus aureus strains after pretreatment of the biological sample by heating 1. Treatment of the sample The biological samples, as prepared in example 2, were treated by heating at 95*C for periods of time ranging from 10 to 30 min, and their OD was determined 10 according to the protocol described in example 2. The OD results are given in table 1 below, which shows that denaturation of the PVL is obtained from 10 min of treatment onward. Table I Heating for Heating for Heating for Strain No. PVL No heating 10 min 20 min 30 min LY990179 + 0.839 2.126 2.76 >3 A960420 + 1.73 2.844 >3 >3 A990438 - 1.545 0.852 0.25 0 A980642 + 0.458 1.364 2.325 >3 LY990301 - 2.123 0.105 0 0 HT20020275 + 0.729 2.269 >3 >3 LY990333 - 0.615 0.202 0 0 HT20040540 + 1.178 >3 >3 >3 15 2. ELISA test The procedure described in example 2, point 2 above was repeated, except for the fact that the culture supernatants of 29 strains (15 PVL+ and 14 PVL-) pretreated at 95*C for 1 h were used. 3. Results 20 The results are shown in figure 2, which is a graph giving the OD of each strain. The results show that all the PVL+ strains are identified by the method of the invention, with an OD level that is sufficiently high to make a distinction with the PVL strains. The pretreatment therefore makes it possible to improve the specificity of the 15 method of the invention. Example 4: Diagnosis of PVL-producing Staphylococcus aureus strains after pretreatment of the biological sample using several types of denaturing treatment 5 1. Treatment of the sample Three clinical PVL+ strains (LY990084, A92007, LUG855) and three clinical PVL- strains (LY990333, LY991321, RN691 1) were used, and were subjected to the following treatments: - no treatment (0), 10 - denaturation by heating at 95*C for 1 h (1), - denaturation with acid, KH 2

PO

4 final concentration of 0.1M, for 30 min on a vortex, then neutralization by adding NaOH (2), - mixed denaturation with acid, KH 2

PO

4 , final concentration of 0.1 M, for 10 min on a vortex, then boiling for 10 min at 95*C, followed by neutralization with 15 NaOH (3). 2. ELISA test The procedure described in example 2 was repeated, except for the fact that the strains pretreated according to point I above and a solution of rabbit antibody 176-89, F(ab'2) fragment labeled with peroxidase, diluted to 1/1000 in PBS-Tween 5% milk 20 were used. 3. Results The results are shown in figure 3, which is a graph giving the OD of each strain. The results show that all the PVL- strains have OD values of between 0.09 and 0.181, whatever the treatment of the sample. 25 For the PVL+ strains, the OD values are higher than those of the PVL- strains after treatment. The increase in OD obtained by chemical treatment, by heat treatment and by a combination of the two is statistically significant (p=0.024, p<0.001, p=0.018, respectively), even though the heat treatment appears to be the most effective under these conditions. 30 The pretreatment therefore makes it possible to improve the specificity of a 16 routine immunological test. Example 5: Demonstration of the early detection of PVL-producing Staphylococcus aureus bacteria using the method of the invention 5 1. Preparation of the biological sample Four clinical strains of S. aureus (2 PVL+ strains and 2 PVL- strains) were precultured according to the procedure described in example 2. The colonies are used to carry out a rich inoculation of 5 ml of CCY medium in a 25 ml glass Erlenmeyer flask. The culture medium is sampled immediately after inoculation, and then successively at 10 1 h, 2 h, 3 h and 18 h after incubation at 37'C with shaking. The culture supernatants are recovered after centrifugation (8000 g, 4'C, 10 min), and are then conserved for a short period at 4'C or at -20'C before use. The samples are treated by heating as described in example 3. 2. ELISA test 15 The procedure described in example 2, point 2 above was repeated, except for the fact that the culture supernatants of the strains pretreated as described in point 1 above were used. 3. Results The OD results are given in table 2, which shows that the PVL can be detected 20 very rapidly with the method of the invention (clear detection from 2 h of culture onward). Table 2 Culture time Strain No. TO Tlh T2h T3h Tl8h A89 0097 PVL + 0.161 1.116 >3 >3 >3 A92 0007 PVL + 0.083 0.975 2.57 >3 >3 LY99 0301 PVL - 0 0 0 0 0 LY990333 PVL- 0 0 0 0 0 Example 6: Demonstration of the early detection of PVL in biological specimens 25 using the method of the invention 1. Preparation of the biological sample The clinical specimens are bronchopulmonary aspirations, bronchoalveolar 17 fluids and pus from skin abscesses or pus from a deep suppuration of patients having S. aureus infections with either PVL+ strains or PVL- strains. These specimens were conserved at -20*C before being analyzed using the method of the invention. After thawing at ambient temperature, the specimens are homogenized by vortexing for 5 15 min. After centrifugation (10 min, at 4000 rpm at 15*C), the supernatant is transferred into an Eppendorf tube, and then denatured for I h at 95'C. For the very viscous samples, the samples are first diluted 50/50 with PBS, pH 7.4, and then vortexed for 15 min. After centrifugation (10 min, at 4000 rpm at 15*C), the supernatant is transferred into an Eppendorf tube, and then denatured for I h 10 at 95'C. In the case of a viscous supernatant, the latter is diluted 50/50 with n-heptane (Merck), and again vortexed for 10 min. After centrifugation (10 min, at 4000 rpm at 15*C), the upper phase corresponding to the n-heptane is completely removed and the lower phase is transferred into another Eppendorf tube in order to be denatured for I h at 95*C. 15 2. ELISA test The procedure described in example 2, point 2 above was repeated, except for the fact that the biological specimens treated as described in point I above were used. 3. Results The PVL assay results are given in tables 3 and 4, which show that the PVL can 20 be detected directly in the biological specimens, whatever the methicillin-sensitivity of the strain, and that there is no false positive (no sample having PVL- strains is detected as containing PVL). Moreover, the pretreatment of the specimen with n-heptane does not impair the detection of the PVL. Table 3 Specimen Type of specimen Specimen Strain* Amount of PVL pretreatment (Pg/ml) 17 Bronchoalveolar lavage no PVL+ <0.05 13 Bronchial aspiration no PVL- <0.05 18 Bronchoalveolar mini lavage no PVL- <0.05 22 Bronchial aspiration no PVL- <0.05 11 Bronchial aspiration no PVL- <0.05 8 Bronchial aspiration PBS + n-heptane PVL- <0.05 16 Bronchial aspiration no PVL- <0.05 10 Bronchial aspiration no PVL- <0.05 18 19 Sputum no PVL- <0.05 14 Bronchial aspiration no PVL- <0.05 15 Bronchial aspiration no PVL- <0.05 12 Bronchial aspiration no PVL- <0.05 7 Bronchial aspiration no PVL- <0.05 5 Bronchial aspiration no PVL- <0.05 23 Joint fluid no PVL- <0.05 20 Peritoneal fluid no PVL- <0.05 4 Drain fluid no PVL+ <0.05 2 Abscess no PVL+ 1 3 Abscess no PVL- <0.05 1 Drain fluid no PVL- <0.05 5 Bronchial aspiration PBS PVL+ 1.2 5 Bronchial aspiration PBS + n-heptane PVL+ 1.2 6 Bronchial aspiration PBS + n-heptane PVL+ 1.2 * The determination of whether the strain was PVL- or PVL+ was carried out by detection of the presence, by PCR, of the LukS-PV and LukF-PV genes encoding PVL within the S. aureus isolates according to the methods described by Vandenesch et al.

2 19 Table 4 Concentration of Detection of mecA Sample Strain* Luk S-PV (pg/ml) gene** Bronchoalveolar lavage PVL+ 0.99 0 Bronchoalveolar lavage PVL+ 1.07 0 Bronchoalveolar lavage PVL+ 20.18 0 Sputum PVL- 0.00 1 Sputum PVL- 0.00 1 Bronchoalveolar lavage PVL- 0.00 1 Sputum PVL- 0.00 1 Bronchoalveolar lavage PVL- 0.00 ND Bronchial aspiration PVL- 0.00 0 Bronchial aspiration PVL- 0.00 0 Bronchial aspiration PVL- 0.00 0 Pneumonia Bronchoalveolar lavage PVL- 0.00 0 Bronchial aspiration PVL- 0.00 1 Bronchial aspiration PVL- 0.00 0 Bronchial aspiration PVL- 0.00 1 Bronchial aspiration PVL- 0.00 0 Bronchial aspiration PVL- 0.00 0 Sputum PVL- 0.00 0 Bronchial aspiration PVL- 0.00 0 Bronchial aspiration PVL- 0.00 0 Bronchial aspiration PVL- 0.00 ND Bronchial aspiration PVL- 0.00 1 Bronchial aspiration PVL- 0.00 0 Skin Abscess PVL+ >1 0 infection Abscess PVL+ 0.42 0 Abscess PVL+ >1 I Abscess PVL+ >1 0 Abscess PVL+ 1.20 0 Abscess PVL+ 1.00 0 Abscess PVL+ 1.30 0 Abscess PVL+ 1.50 1 Abscess PVL+ 1.82 0 Abscess PVL+ >2 0 Abscess PVL+ 0.92 0 Abscess PVL+ >2 0 Abscess PVL+ 0.27 0 Abscess PVL+ >2 0 Abscess PVL+ 1.72 0 Abscess PVL+ 1.41 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 1 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 20 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 1 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 Abscess PVL- 0.00 0 * The determination of whether the strain was PVL- or PVL+ was carried out by detection of the presence, by PCR, of the LukS-PV and LukF-PV genes encoding PVL within the S. aureus isolates according to the methods described by Vandenesch et al.

2 ** The detection of the mecA gene encoding the methicillin resistance within the 5 S. aureus isolates was carried out according to the methods described by Vandenesch et al.

2 21 Literature 1: Ward PD, Turner WD, 1980, Infect. Immun., 28(2): 393-397 2: Vandenesch et al., 2003, Fmerg Infect Dis, 9(8): 978-984 3: Dufour P et al., 2002, Clin infect Dis, 35: 819-824 4: Cribier B et al, 1992, Dermatology, 185: 175-185 5: Freney J et al., 2000, Pricis de Bacteriologie Clinique [Handbook of clinical bacteriology], 40: 298 and 793-794 6: J. Histochem. Cytochem. 45: 481-491, 1997 7: Labandeira-Rey M et al, Science 2007, in press The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. it is not suggested or represented that any or all of these matters formed part of the prior art base or were comnion general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. Where the terms "Comprise", "compises", "comprised" or "corriprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features. integers, steps or components, but not precluding the presence of one or more other features, integers. steps or components, or group thereof.

Claims (8)

1. A method for in vitro diagnosis of Staphylococcu.s aureus producing Panton Valentine Leukocidin (PVL) using a biological sample derived from an individual liable to be colonized by or infected with Staphylococcus aureus, wherein the diagnosis is carried out by detection of PV., using a routine immunological test chosen from an enzyme-linked immunosorbent assay (ELISA), lateral flow immunochromatography and an agglutination test and wherein said biological sample is pretreated in order to denature the PVL, the pretreatment comprising heating at a temperature of between 60 and 100*C for a period of at least 10 min or using an acid.

2. The method as claimed in claim 1, wherein the routine immunological test is a sandwich method.

3. The method as claimed in claim 1 or 2. wherein the immunological test uses at least one anti-PVL monoclonal antibody.

4. The method as claimed in any one of claims I to 3, wherein it comprises an additional step consisting in determining whether the Staphylococcus aureus present in said biological sample are methicillin-resistant (MRSA) or methicillin-sensitive (MSSA) bacteria.

5. The method as claimed in any one of claims 1 to 4, wherein the pretreatment comprises heating at a temperature of be tween 60 and 100C for a period of at least 20 min or more.

6. The method as claimed in any one of claims 1 to 5, wherein the pretreatment comprises heating at a temperature of between 60 and 100*C for a period of from 10 min to 30 min.

7. The method as clairned in any one of claims 1 to 5, wherein. the pretreatment comprises the use of KH2PO 4 as an acid.

8. A. method according to claim 1, substantially as hereinbefore described with reference to the Examples and/or Figures.